1. Field of the Invention
The present invention relates to a base plate and a method for immunoassay of thyroxine using fluorescent particles.
2. Description of the Related Art
Fluorescence detection methods have been hitherto used in a large variety of fields, as analytic methods that are highly sensitive and simple and are capable of quantitatively determining proteins, enzymes, inorganic compounds and the like. These fluorescence detection methods are methods by which a sample which is suspected to contain a substance to be detected (test substance) that is excited by light having a specific wavelength and emits fluorescence, is irradiated with excitation light having the specific wavelength, fluorescence is detected at that time, and thereby the presence of the test substance is verified. Furthermore, in the case where the substance is not a fluorescent material, a method of bringing a substance which specifically binds to a test substance labeled with a fluorescent dye, into contact with a sample, subsequently detecting fluorescence in the same manner as described above, and thereby verifying the presence of this binding, that is, the presence of the test substance, is also widely used.
In regard to such fluorescent detection methods, a method of utilizing the effect of electric field reinforcement by plasmon resonance so as to increase the sensitivity of detection, is known. In such a method, in order to induce plasmon resonance, a sensor chip in which a metal layer is provided in a predetermined region on a transparent support is prepared, and excitation light is caused to enter the sensor chip through the surface of the support on the opposite side of the metal layer-formed surface with respect to the interface between the support and the metal film, at a predetermined angle that is greater than or equal to the total reflection angle. Irradiation of such excitation light causes generation of surface plasmon at the metal layer. Through the electric field reinforcing action brought by the generation of such surface plasmon, fluorescence is reinforced, and thereby the signal/noise ratio (S/N ratio) is increased. In a fluorescence detection method involving surface plasmon excitation (hereinafter, referred to as “SPF method”), the degree of signal reinforcement can be about 10 times, and analysis can be conducted with high sensitivity, as compared to a fluorescence detection method involving epi-illumination excitation (also called epi-illumination fluorescence method).
For example, in the light signal detection method for determining the amount of a test substance as described in JP 2010-190880 A, a sensor chip which has a sensor unit including a metal layer, provided in a predetermined region of one surface of a dielectric plate is prepared, and the sensor unit of the sensor chip is brought into contact with a sample. Through such contact, a binding substance attached with a photoresponsive labeling substance in an amount equivalent to the amount of the test substance contained in the sample, binds to the sensor unit. Subsequently, excitation light is irradiated to the predetermined region, and light from the photoresponsive labeling substance, which is produced in the electric field-reinforced field produced on the metal layer, is detected. Thus, the amount of the test substance can be determined. Furthermore, in this method, it is also possible to use, as a photoresponsive labeling substance, a light transmitting material which transmits the light produced from a photoresponsive substance, and in which plural photoresponsive substances are included such that the metal quenching occurring when the photoresponsive substances approach the metal layer is prevented.
Thyroxine (T4; also called thyroxin) is one kind of thyroid hormones secreted from the thyroid gland. Generally, thyroxine acts on cells of the whole body and has a function of increasing the metabolic rate of cells. Known examples of thyroid hormones include thyroxine (T4) and triiodothyronine (T3), but a majority of the thyroid hormones circulating in the blood is thyroxine (T4). As a method for measuring this thyroxine, an enzymatic immunoassay method which involves simple operation has been conventionally known. For example, JP 1995-146293 A (JP-H-07-146293 A) discloses an enzymatic immunoassay method utilizing a competition method for measuring the amount of thyroxine, and describes an enzymatic immunoassay method using magnetic microbeads for the purpose of shortening the enzymatic reaction time. Furthermore, JP 1994-094709 A (JP-H-06-094709 A) discloses an enzymatic immunoassay method and a reagent for measuring free thyroxine as a free thyroid hormone, and describes an enzymatic immunoassay method and a reagent which us a specific labeled enzyme for the purpose of suppressing the influence of albumin in blood.